CN104012520A - Anti-oxidative cattle frozen semen diluent and preparation method thereof - Google Patents
Anti-oxidative cattle frozen semen diluent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses anti-oxidative cattle frozen semen diluent. The anti-oxidative cattle frozen semen diluent is composed of the following raw materials by mass: 20-26 parts of trihydroxy methyl-aminomethane, 10-15 parts of trisodium citrate, 7-15 parts of fructose, 700-800 parts of distilled water, 230-260 parts of fresh yolk, 0.125-0.375 part of sodium selenite, 2-32 parts of Vitamin E, 90-95 parts of glycerol, 1-2 parts of penicillin potassium for injection (specification of 1.6 million units/g) and 1-2 parts of streptomycin sulfate for injection (specification of 1.0 million units/g). The preparation method of the anti-oxidative cattle frozen semen diluent comprises the steps of preparing a basic buffer solution, a basic diluent and a semen diluent; then transferring the prepared semen diluent to a condition of 4 DEG C, and balancing for 5-20 min to obtain the anti-oxidative cattle frozen semen diluent. Compared with a conventional anti-oxidative cattle frozen semen diluent, the anti-oxidative cattle frozen semen diluent provided by the invention increases semen motility rate by 24.56%, increases 4 h semen motility rate by 12.39%, increases semen perforatorium integrity rate by 38.72%, increases GSH-PX activity by 260%, and reduces semen aberration rate by 5.49%.
Description
Technical field
The invention belongs to animal frozen semen production technology, be specifically related to a kind of preparation method of the cold extender of freezing semen that adds trace elements of selenium and vitamin E.
Background technology
The breeding of ox is an important step in cowboying production process, and the increase of cows quantity, the raising of quality all must realize by breeding this process.Artificial insemination and embryo transfer technology are twice great technological revolutions during cattle-raising is produced, and can give full play to the breeding potential of outstanding stock bull and cow, demonstrate good application prospect.The prerequisite of artificial insemination and embryo transfer technology application is the application of the Techniques of preserving of ox seminal fluid.The success of semen cryopreservation is a significant innovation on technology of artificial insemination, it has not only solved the long-term problem of preserving of seminal fluid, make the application of seminal fluid not be subject to the restriction of time, region and breeding stock life, and improve the availability of good male animal, accelerate the incubation of kind and the paces of improvement.One of main poultry kind that during ox produces as livestock breeding, natural propagation rate is minimum, using artificial insemination is more great with the meaning that embryo transfer technology improves ox fertility.
Bull semen is in the time of freezing preservation; seminal plasma is limited to the protective capability of sperm; therefore; the freezing preservation of carrying out seminal fluid will be used in conjunction with dilution; farthest protect sperm; thereby dilution highlights its importance in semen cryopreservation, become the principal element that ensures semen quality.Artificial insemination has been a ripe technology, but while using frozen semen to carry out artificial insemination, its fertilization effect is lower than using the effect of fresh semen, its reason is that fresh semen is in the time making frozen semen, at it, mass change freezing and sperm while thawing, is to affect the successful key factor of artificial insemination.
Existing freezing seminal fluid dilution normally adds thinner, nutritional agents, antibacterial, freeze proof, anti-shock and buffering protectant etc.; and to seminal fluid in freezing and course of defrosting can anti-oxidative damage material; be confined on the materials such as vitamin E, vitamin C and glutathione; due to anti-oxidative damage scarce capacity; reduce motility rate, survival rate and acrosomal integrity etc. after thawing, reduced semen quality.
Summary of the invention
The present invention be directed to the present situation of the oxidation resistance deficiency of existing freezing seminal fluid dilution, a kind of oxidation resistant cattle freezing seminal fluid dilution is provided, it is on the basis of the constituent conventional at freezing seminal fluid dilution, certain density trace elements of selenium and vitamin E are added, improve the oxidation resistance of freezing seminal fluid dilution, improve sperm viability and acrosomal integrity, extended sperm life span, improved the object of frozen semen quality.Another object of the present invention is to provide the preparation method of oxidation resistant cattle freezing seminal fluid dilution.
The present invention is achieved by the following technical solutions.
Oxidation resistant cattle freezing seminal fluid dilution of the present invention, comprise the raw material composition of following mass parts: trishydroxymethylaminomethane 20 ~ 26, trisodium citrate 10 ~ 15, fructose 7 ~ 15, distilled water 700 ~ 800, fresh yolk 230 ~ 260, sodium selenite 0.125 ~ 0.375, vitamin E2 ~ 32, glycerine 90-95, (specification is that (specification is 1,000,000 units/g) 1 ~ 2 for 1,600,000 units/g) 1 ~ 2, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Oxidation resistant cattle freezing seminal fluid dilution preparation method of the present invention, comprises the steps:
(1) after preparing basic buffer solution and taking 20 ~ 26 parts of trishydroxymethylaminomethanes, 10 ~ 15 parts of trisodium citrates, 7 ~ 15 parts of fructose and mix, add in 700 ~ 800 parts of distilled water and stir, for subsequent use:
(2) preparing basic dilution takes the basic buffer solution that 230 ~ 260 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 ~ 2 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1 ~ 2 part again, stir, for subsequent use;
(3) prepare sodium selenite that semen diluent takes 0.125 ~ 0.375 part, the vitamin E of 2 ~ 32 parts and add in the basic dilution that (2) prepare, after stirring, take again 90-95 part glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
Oxidation resistant cattle freezing seminal fluid dilution of the present invention, the raw material composition of preferred following mass parts: trishydroxymethylaminomethane 22-24 part, trisodium citrate 12-13 part, fructose 10-12 part, distilled water 750-780 part, fresh yolk 240-250 part, sodium selenite 0.25-0.3 part, vitamin E2 0-25 part, glycerine 92-94 part, (specification is that (specification is 1,000,000 units/g) 1-1.5 part for 1,600,000 units/g) 1.5-2 part, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Oxidation resistant cattle freezing seminal fluid dilution preparation method of the present invention, comprises the steps:
(1) after preparing basic buffer solution and getting 22 ~ 24 parts of trishydroxymethylaminomethanes, 12 ~ 13 parts of trisodium citrates, 10 ~ 12 parts of fructose and mix, add in 750 ~ 780 parts of distilled water and stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 240 ~ 250 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 ~ 1.5 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1.5 ~ 2 parts again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0. 25 ~ 0.3 part, the vitamin E of 20 ~ 25 parts and add in the basic dilution that (2) prepare, after stirring, get again 92-94 part glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
The sperm of ox is a cell that height breaks up, has activity and contains genetic material, and there is the film of lipoprotein character on plasmalemmae of sperms surface.Head is mainly made up of cell nucleus, wherein mainly containing compositions such as nucleoprotein and DNA, RNA.Being covered by acrosome above of core.Acrosome is double-deck film capsule, contains sperm neutral proteinase, hyaluronidase etc., and these enzymes are all relevant with fertilization.Acrosome rather unstable, easily sex change and coming off from the head, can make the fertility reduction of sperm or completely lost.In the time producing frozen semen, must carry out seminal fluid freezing, using the frozen semen that must thaw when frozen semen.Sperm produces a large amount of active oxygens (ROS) in freezing and course of defrosting, and active oxygen can be oxidized unsaturated fatty acid.On mitochondrial membrane in sperm membrane and sperm, be rich in unsaturated fatty acid, very responsive to lipid peroxidation, excessive active oxygen and membrane phospholipid peroxidating can cause cryopreservation sperm vigor to reduce.Be embodied in sperm tail stage casing and principal piece after birth breaks, axial filament exposes, abnormalities.Mitochondria is the place that energy substance oxidative metabolism discharges adenosine triphosphate (ATP), there is extremely certainly will causing dysfunction in its structure, thereby the metabolism that causes the energy substance in dilution is obstructed, ATP produces not enough, the motion of sperm is affected like this, the ability that final sperm trip is broken through egg membrane to ovum reduces, and cannot complete normal fertilization process.So frozen semen quality declines, with frozen semen, conception rate is not high and very unstable.
Active oxygen belongs to free radical, and free radical produces in metabolic process in vivo.In sperm, the radical pair sperm of suitable number is harmless, and they are participating in the processes such as signal transmission.Once free radical exceeds the removing ability of itself, can cause body oxidative damage.The infringement of radical pair sperm mainly makes lipid peroxidation and destroys cell membrane and endomembrane system, plasmalemmae of sperms will be followed the string by the attack of free radical, to protein sulfhydryl, methyl mercapto or trp residue reaction, cause protein molecule polymerization or crosslinked, cause the forfeiture of protein function or enzymic activity; Destroy nucleic acid structure, attack purine and pyrimidine base, cause change and the loss of base, make abnormal gene expression, occur sudden change, thereby cause that spermatogenesis systematicness is disorderly, occur old and feeble.This life of sperm has balance free radical and removes the ability of unnecessary free radical, and in sperm, radicals scavenging system comprises the first line of defence of enzyme composition: as superoxide dismutase (SOD), glutathione peroxidase (GSH-P
x), second defence line is made up of low molecule scavenger: as ubiquinone
10, glutathione, vitamin E, carotin, cysteine, three lines of defence is that the reparation that utilizes DNA and protein, reduction, the recovery of mucous membrane ATP and the minimizing of intracellular calcium of protein sulfhydryl carry out antagonism free radical.
The present invention, at cattle freezing seminal fluid dilution, except the component of routine application, has added certain density trace elements of selenium.
Selenium is the essential composition of some antioxidases such as glutathione peroxidase, and it,, by eliminating the MDA having produced on plasmalemmae of sperms and mitochondrial membrane, blocks the pathogenic effects of reactive oxygen species and free radicals.
The present invention has also added vitamin E in cattle freezing seminal fluid dilution.Vitamin E is important fat-soluble blocking-up type antioxidant in body, in plasmalemmae of sperms, the peroxidization of lipid is the chain reaction of free radical, its consequence is that free radical is on the increase, and peroxidization is constantly accelerated, and finally makes fatty acid chain rupture and destroys plasmalemmae of sperms, interior membrane structure.Vitamin E energy Mulberry Extract makes phenolic group on its chromene ring lose a hydrogen atom and form vitamin e free radical; then further react and form non-free radical product fertility enormous legendary fish, which could change into a roc with another free radical; and prevented carrying out continuously of lipid chain reaction, protect plasmalemmae of sperms and inner membrance.After vitamin E and radical reaction, own vitamin e hydroxy radical, i.e. the oxidized form vitamin E of being oxidized to itself.
Selenium and vitamin E are both antioxidant, but its Present site is different with the mode of action, and vitamin E is present in cell membrane, by closing with free-fat root knot, prevent in fatty acid metabolism and produce peroxide, prevent its infringement to lipid film.Selenium is present in cell sap, and in the time that vitamin E can not stop the peroxide that too much unsaturated fatty acid forms completely, selenium can be by strengthening the activity of GSH-PX, and performance destroys the effect of peroxide, and both can not replace.Having data to prove that selenium and vitamin E are worked in coordination with is greater than both and acts on respectively sum improving the effect of bringing into play aspect body antibody horizontal and the infringement of control radical pair body.In the time that selenium and vitamin E addition are not suitable for, especially between the anti-oxidant requirement of selenium and poisoning limiting quantity, difference is little, easily causes selenium excessive poisoning, and therefore, the consumption of sodium selenite of the present invention is the parameter obtaining through years of researches.
The oxidation resistant cattle freezing seminal fluid dilution of the present invention is tested compared with the prior art
1. test method
1.1 establish 4 processing taking the conventional applied component (being trishydroxymethylaminomethane, trisodium citrate, fructose, distilled water, fresh yolk, glycerine, injection benzylpenicillin potassium and streptomycin sulphate for injection) of cattle freezing seminal fluid dilution as contrast 1, on contrast component basis, add vitamin E for contrast 2, add sodium selenite for contrast 3, adding vitamin E and sodium selenite is of the present invention group.The mass parts of said components is respectively: trishydroxymethylaminomethane 24, trisodium citrate 12, fructose 10, distilled water 750, fresh yolk 250, glycerine 90, (specification is 1,600,000 units/g) 1 to injection benzylpenicillin potassium, (specification is 1,000,000 units/g) 1 to streptomycin sulphate for injection, vitamin E2 0, sodium selenite 0.25.
Randomly draw 4 stock bull compositions for 1.2 every groups, the former seminal fluid after semen collection is made frozen semen according to conventional method dilution, balance, packing etc.
The step that the cattle freezing seminal fluid dilution preparation method of 1.3 4 processing has described with summary of the invention, just the raw material components difference of each processing.
The mensuration of 1.4 semen qualities
1.4.1 the inspection of sperm motility rate and time-to-live: after thawing, seminal fluid checks motility rate immediately, checks motility rate (time-to-live) after then 37 DEG C of insulating boxs are preserved 4h.
1.4.2 Sperm acrasomal integvity inspection: adopt National Standard Method to measure.
1.4.3 the inspection of rate of teratosperm: adopt National Standard Method to measure.
1.4.4 the integrity checking to plasmalemmae of sperms: hypotonic expansion method (HOST).
2. statistical analysis
Variance analysis and the DuncanShi method of test data application SAS9.0 statistical analysis software compare.
3. result of the test
Result of the test is in table 1.
Of the present invention group of table 1 and the impact of control group freezing seminal fluid dilution on semen quality
| Test grouping | Sample number | Sperm motility rate (%) | 4h sperm motility rate (%) | Rate of teratosperm (%) | Sperm acrasomal integvity (%) | The activity (U/L) of GSH-PX |
| Contrast 1 group | 36 | 22.80 C | 8.50 C | 13.10 A | 33.58 D | 645.07 c |
| Contrast 2 groups | 36 | 31.57 B | 13.23 B | 10.76 B | 41.94 C | 864.92 C |
| Contrast 3 groups | 36 | 33.16 B | 15.12 B | 9.79 B | 62.96 B | 901.29 B |
| Of the present invention group | 36 | 47.36 A | 20.89 A | 7.61 C | 72.30 A | 2322.09 A |
Note: same column subscript indicates adjacent letters and represents significant difference (P < 0.05), indicates extremely significantly (P < 0.01) of alternate letter representation difference.
Result of the test shows, in the conventional applied component of cattle freezing seminal fluid dilution, add separately after vitamin E (contrasting 2 groups) and sodium selenite (contrasting 3 groups), the specific activity contrast 1 of sperm motility rate, 4h sperm motility rate, Sperm acrasomal integvity, GSH-PX all has raising extremely significantly, rate of teratosperm is decline extremely significantly, illustrates that interpolation vitamin E effect is remarkable separately; Wherein, adding separately sodium selenite (contrasting 3 groups) has extremely significantly and improves than the Sperm acrasomal integvity that interpolation vitamin E (contrasting 2 groups) is processed separately, the activity of GSH-PX significantly improves, and illustrates that sodium selenite is better than vitamin E to improving the quality of cattle freezing seminal fluid dilution.But, the present invention is processed with interpolation vitamin E (contrasting 2 groups) and sodium selenite (contrasting 3 groups) are found more afterwards separately, the specific activity that the present invention processes sperm motility rate, the 4h sperm motility rate of adding more separately vitamin E (contrast 2 groups) and sodium selenite (contrasting 3 groups), Sperm acrasomal integvity, GSH-PX contrasts 1 all raising extremely significantly, rate of teratosperm is decline extremely significantly, illustrate vitaminize E and sodium selenite to the improvement of cattle freezing seminal fluid dilution quality except the effect of self, between the two also have interaction.
The invention has the beneficial effects as follows: the present invention has added sodium selenite and vitamin E in dilution, significantly improve the activity of motility rate, Sperm acrasomal integvity, GSH-PX after thawing of semen compared with the prior art utmost point, therefore, effectively eliminate the infringement of active oxygen to sperm, effectively protect the integrality of plasmalemmae of sperms, show by the result of hypotonic expansion experiment, this dilution can effectively be protected the integrality of plasmalemmae of sperms; Meanwhile, rate of teratosperm extremely significantly declines compared with prior art.Through comparative test, the present invention has improved 24.56 percentage points compared with the sperm motility rate of the cattle freezing seminal fluid dilution of prior art, 4h sperm motility rate has improved 12.39 percentage points, Sperm acrasomal integvity has improved 38.72 percentage points, the activity of GSH-PX has improved 260%, and rate of teratosperm has declined 5.49 percentage points.
Embodiment
embodiment 1
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: 20 parts of trishydroxymethylaminomethanes, 10 parts of trisodium citrates, 7 parts, fructose, 700 parts of distilled water, 230 parts, fresh yolk, 0.125 part of sodium selenite, vitamin E2 part, 90 parts of glycerine, (specification is that (specification is 1,000,000 units/g) 1 part for 1,600,000 units/g) 1 part, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 20 parts of trishydroxymethylaminomethanes, 10 parts of trisodium citrates, 7 parts of fructose and mix, add 700 parts of distilled water to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 230 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1 part again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.125 part, the vitamin E of 2 parts and add in the basic dilution that (2) prepare, after stirring, get again 90 parts of glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
embodiment 2
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: 26 parts of trishydroxymethylaminomethanes, 15 parts of trisodium citrates, 15 parts, fructose, 800 parts of distilled water, 260 parts, fresh yolk, 0.375 part of sodium selenite, 32 parts of vitamin Es, 95 parts of glycerine, (specification is that (specification is 1,000,000 units/g) 2 parts for 1,600,000 units/g) 2 parts, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 26 parts of trishydroxymethylaminomethanes, 15 parts of trisodium citrates, 15 parts of fructose and mix, add 800 parts of distilled water to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 260 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 2 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 2 parts again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.375 part, the vitamin E of 32 parts and add in the basic dilution that (2) prepare, after stirring, get again 95 parts of glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
embodiment 3
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: 24 parts of trishydroxymethylaminomethanes, 12 parts of trisodium citrates, 10 parts, fructose, 750 parts of distilled water, 240 parts, fresh yolk, 0.25 part of sodium selenite, 5 parts of vitamin e1s, 92 parts of glycerine, (specification is that (specification is 1,000,000 units/g) 1.5 parts for 1,600,000 units/g) 1.5 parts, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 24 parts of trishydroxymethylaminomethanes, 12 parts of trisodium citrates, 10 parts of fructose and mix, add 750 parts of distilled water to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 240 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1.5 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1.5 parts again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.25 part, the vitamin E of 15 parts and add in the basic dilution that (2) prepare, after stirring, get again 92 parts of glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
embodiment 4
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: 20 parts of trishydroxymethylaminomethanes, 15 parts of trisodium citrates, 7 parts, fructose, 800 parts of distilled water, 230 parts, fresh yolk, 0.375 part of sodium selenite, vitamin E2 part, 95 parts of glycerine, (specification is that (specification is 1,000,000 units/g) 2 parts for 1,600,000 units/g) 1 part, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 20 parts of trishydroxymethylaminomethanes, 15 parts of trisodium citrates, 7 parts of fructose and mix, add 800 parts of distilled water to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 230 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 2 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1 part again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.375 part, the vitamin E of 2 parts and add in the basic dilution that (2) prepare, after stirring, get again 95 parts of glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
embodiment 5
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: 26 parts of trishydroxymethylaminomethanes, 10 parts of trisodium citrates, 15 parts, fructose, 700 parts of distilled water, 260 parts, fresh yolk, 0.125 part of sodium selenite, 32 parts of vitamin Es, 90 parts of glycerine, (specification is that (specification is 1,000,000 units/g) 1 part for 1,600,000 units/g) 2 parts, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 26 parts of trishydroxymethylaminomethanes, 10 parts of trisodium citrates, 15 parts of fructose and mix, add 700 parts of distilled water to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 260 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 2 parts again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.125 part, the vitamin E of 32 parts and add in the basic dilution that (2) prepare, after stirring, get again 90 parts of glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
embodiment 6
Oxidation resistant cattle freezing seminal fluid dilution, raw material by following mass parts forms: trishydroxymethylaminomethane 22-24 part, trisodium citrate 12-13 part, fructose 10-12 part, distilled water 750-780 part, fresh yolk 240-250 part, sodium selenite 0.25-0.3 part, vitamin E2 0-25 part, glycerine 92-94 part, (specification is that (specification is 1,000,000 units/g) 1-1.5 part for 1,600,000 units/g) 1.5-2 part, streptomycin sulphate for injection to injection benzylpenicillin potassium.
Prepare oxidation resistant cattle freezing seminal fluid dilution step as follows:
(1) after preparing basic buffer solution and getting 22-24 part trishydroxymethylaminomethane, 12-13 part trisodium citrate, 10-12 part fructose and mix, add distilled water 750-780 part to stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that the fresh yolk of 240-250 part prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) for 1,600,000 units/g) and 1-1.5 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1.5-2 part again, stir, for subsequent use;
(3) prepare semen diluent and get the vitamin E of the sodium selenite of 0.25-0.3 part, 20-25 part and add in the basic dilution that (2) prepare, after stirring, get again 92-94 part glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
Claims (2)
1. an oxidation resistant cattle freezing seminal fluid dilution, it is characterized in that, oxidation resistant cattle freezing seminal fluid dilution comprises the raw material composition of following mass parts: trishydroxymethylaminomethane 20 ~ 26, trisodium citrate 10 ~ 15, fructose 7 ~ 15, distilled water 700 ~ 800, fresh yolk 230 ~ 260, sodium selenite 0.125 ~ 0.375, vitamin E2 ~ 32, glycerine 90-95, (specification is that (specification is 1,000,000 units/g) 1 ~ 2 for 1,600,000 units/g) 1 ~ 2, streptomycin sulphate for injection to injection benzylpenicillin potassium;
Oxidation resistant cattle freezing seminal fluid dilution preparation method, comprises the steps:
(1) after preparing basic buffer solution and getting 20 ~ 26 parts of trishydroxymethylaminomethanes, 10 ~ 15 parts of trisodium citrates, 7 ~ 15 parts of fructose and mix, add in 700 ~ 800 parts of distilled water and stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 230 ~ 260 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 ~ 2 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1 ~ 2 part again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0.125 ~ 0.375 part, the vitamin E of 2 ~ 32 parts and add in the basic dilution that (2) prepare, after stirring, get again 90-95 part glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
2. an oxidation resistant cattle freezing seminal fluid dilution, it is characterized in that, oxidation resistant cattle freezing seminal fluid dilution comprises the raw material composition of following mass parts: trishydroxymethylaminomethane 22-24 part, trisodium citrate 12-13 part, fructose 10-12 part, distilled water 750-780 part, fresh yolk 240-250 part, sodium selenite 0.25-0.3 part, vitamin E2 0-25 part, glycerine 92-94 part, (specification is that (specification is 1,000,000 units/g) 1-1.5 part for 1,600,000 units/g) 1.5-2 part, streptomycin sulphate for injection to injection benzylpenicillin potassium;
Oxidation resistant cattle freezing seminal fluid dilution preparation method, comprises the steps:
(1) after preparing basic buffer solution and getting 22 ~ 24 parts of trishydroxymethylaminomethanes, 12 ~ 13 parts of trisodium citrates, 10 ~ 12 parts of fructose and mix, add in 750 ~ 780 parts of distilled water and stir, for subsequent use:
(2) preparing basic dilution gets the basic buffer solution that 240 ~ 250 parts of fresh yolk are prepared with (1) and evenly mixes, (specification is that (specification is 1,000,000 units/g) to 1,600,000 units/g) He 1 ~ 1.5 part streptomycin sulphate for injection to add the injection benzylpenicillin potassium of 1.5 ~ 2 parts again, stir, for subsequent use;
(3) prepare semen diluent and get the sodium selenite of 0. 25 ~ 0.3 part, the vitamin E of 20 ~ 25 parts and add in the basic dilution that (2) prepare, after stirring, get again 92-94 part glycerine and add basic dilution, mix, for subsequent use;
(4) semen diluent (3) being prepared moves to balance 5-20min under 4 DEG C of conditions, makes oxidation resistant cattle freezing seminal fluid dilution.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105010306A (en) * | 2015-06-30 | 2015-11-04 | 阜阳师范学院 | Fox semen diluting liquid |
| CN106190955A (en) * | 2016-07-27 | 2016-12-07 | 柳州市柳南区安顺养殖协会 | A kind of method improving cow frozen semen Sperm motility and external fertilization rate |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110463687A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of the preservation liquid and store method of sheep sperm |
| CN112640886A (en) * | 2020-12-18 | 2021-04-13 | 洛阳市洛瑞牧业有限公司 | Cattle frozen semen diluent and preparation method thereof |
| CN112931483A (en) * | 2021-01-29 | 2021-06-11 | 海南晨海水产有限公司 | Decapterus maruadsi sperm freezing liquid capable of keeping high survival and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105010306A (en) * | 2015-06-30 | 2015-11-04 | 阜阳师范学院 | Fox semen diluting liquid |
| CN106190955A (en) * | 2016-07-27 | 2016-12-07 | 柳州市柳南区安顺养殖协会 | A kind of method improving cow frozen semen Sperm motility and external fertilization rate |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110463687A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of the preservation liquid and store method of sheep sperm |
| CN110463688B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Diluent of sheep semen and preservation method |
| CN110463687B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Preservation solution and preservation method for sheep semen solution |
| CN112640886A (en) * | 2020-12-18 | 2021-04-13 | 洛阳市洛瑞牧业有限公司 | Cattle frozen semen diluent and preparation method thereof |
| CN112931483A (en) * | 2021-01-29 | 2021-06-11 | 海南晨海水产有限公司 | Decapterus maruadsi sperm freezing liquid capable of keeping high survival and preparation method thereof |
| CN112931483B (en) * | 2021-01-29 | 2022-05-24 | 海南晨海水产有限公司 | Decapterus maruadsi sperm freezing liquid capable of keeping high survival and preparation method thereof |
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